I selected stable HEK293 clones with 0.4mg/mL Geneticin, now I am expanding them. Do I have to keep the same selective pressure or can I decrease the antibiotic concentration?
In my opinion, it is completely unnecessary to add antibiotic to the growth medium if you construct has been integrated into the genome and transcriptionally active
HEK293 is very sensitive cells with low adhesion affinity and proliferation rate. I recommend decrease antibiotic concentration and increase serum to %20 of complete medium (RPMI) in passages. In other hand you wouldn't need to keep same concentration if your stable cells has been selected previously.
Selection with Geneticin or G418 can be a bit tricky since it often kills cells slowly. I would recommend passing the cells at least 3 times under selective pressure before eliminating the antibiotic. Also note that some 293 strains, such as 293T cells, are already G418-resistant. The parental HEK cell line should be fine for your purposes.
You can reduce the selection pressure now, because once it is a stable cell line you no longer need the selection pressure, still i prefer to keep it in low selection pressure.
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